U.S. patent application number 10/923635 was filed with the patent office on 2005-02-24 for high molar succinate yield bacteria by increasing the intracellular nadh availability.
Invention is credited to Bennett, George N., San, Ka-Yiu, Sanchez, Ailen.
Application Number | 20050042736 10/923635 |
Document ID | / |
Family ID | 34198245 |
Filed Date | 2005-02-24 |
United States Patent
Application |
20050042736 |
Kind Code |
A1 |
San, Ka-Yiu ; et
al. |
February 24, 2005 |
High molar succinate yield bacteria by increasing the intracellular
NADH availability
Abstract
The invention relates to increasing the yield of succinate in
bacteria by increasing the intracellular availability of cofactors
such as NADH.
Inventors: |
San, Ka-Yiu; (Houston,
TX) ; Bennett, George N.; (Houston, TX) ;
Sanchez, Ailen; (Houston, TX) |
Correspondence
Address: |
JENKENS & GILCHRIST
1401 MCKINNEY
SUITE 2600
HOUSTON
TX
77010
US
|
Family ID: |
34198245 |
Appl. No.: |
10/923635 |
Filed: |
August 20, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60497195 |
Aug 22, 2003 |
|
|
|
Current U.S.
Class: |
435/89 ;
435/455 |
Current CPC
Class: |
C12P 7/46 20130101 |
Class at
Publication: |
435/089 ;
435/455 |
International
Class: |
C12P 019/30; C12N
015/85; C12P 007/40 |
Goverment Interests
[0002] The present invention has been developed with funds from the
National Science Foundation. Therefore, the United States
Government may have certain rights in the invention.
Claims
What is claimed is:
1. A method of increasing the production of succinate within a
cell, comprising the steps of, mutating one or more genes that
encode enzymes involved in metabolic reactions that consume NADH;
transforming the mutant strain with one or more expression plasmids
encoding enzymes that produce NADH; and increasing intracellular
levels of NADH.
2. The method of claim 1, wherein the enzymes involved in metabolic
reactions that consume NADH are selected from the group consisting
of lactate dehydrogenase, alcohol dehydrogenase, pyruvate form ate
lyase, glucose permease, formate dehydrogenase and combinations
thereof.
3. The method of claim 1, wherein the expression plasmid contains
the gene encoding pyruvate carboxylase.
4. The method of claim 1, wherein the expression plasmid contains
the gene encoding NAD-dependent formate dehydrogenase.
5. A method of increasing the NADH flux in a cell, comprising the
step of mutating one or more genes that encode enzymes involved in
metabolic reactions, wherein said mutations result in increased
intracellular levels of NADH.
6. The method of claim 5, wherein the enzymes are selected from the
group consisting of lactate dehydrogenase, alcohol dehydrogenase,
pyruvate formate lyase, glucose permease, formate dehydrogenase and
combinations thereof
7. The method of claim 5, where the cell is transformed with a
plasmid that expresses pyruvate carboxylase.
8. The method of claim 5, where the cell is transformed with a
plasmid that expresses NAD-dependent formate dehydrogenase.
9. A method for the biosynthesis of a target compound comprising
the steps of increasing the intracellular levels of NADH, and
directing the increased NADH levels towards the biosynthesis of
said one or more target compounds.
10. The method of claim 9, wherein the intracellular levels of NADH
are increased by the mutation of one or more genes that encode for
NADH-utilizing enzymes.
11. The method of claim 9, wherein the intracellular levels of
A-CoA are increased by deletion of one or more NADH utilizing
pathways.
12. The method of claim 9 wherein said target compound is
succinate.
13. A microorganism which contains one or more mutant genes,
wherein said microorganism displays increased yields of
succinate.
14. The microorganism of claim 13, wherein said one or more genes
encode enzymes that are involved in metabolic pathways that utilize
NADH.
15. The microorganism of claim 14, wherein the enzymes are selected
from the group consisting of lactate dehydrogenase, alcohol
dehydrogenase, pyruvate formate lyase, glucose permease, formate
dehydrogenase and combinations thereof.
16. The microorganism of claim 13, wherein the molar ratio of the
succinate yield to glucose substrate is greater than 1.0.
17. The microorganism of claim 13, wherein the molar ratio of the
succinate yield to glucose substrate is at least about 1.3.
18. The microorganism of claim 13, wherein the molar ratio of the
succinate yield to glucose substrate is greater than about 1.3.
Description
PRIOR RELATED APPLICATIONS
[0001] This application claims the benefit of U.S. provisional
application No. 60/497,195, filed Aug. 22, 2003.
REFERENCE TO MICROFICHE APPENDIX
[0003] Not applicable.
FIELD OF THE INVENTION
[0004] The invention relates to increasing the yield of succinate
in bacteria by increasing the intracellular availability of
cofactors such as NADH.
BACKGROUND OF THE INVENTION
[0005] Succinic acid has drawn much interest because it has been
used as a precursor of numerous chemicals including pharmaceuticals
and biodegradable polymers. Succinic acid is a member of the
C.sub.4-dicarboxylic acid family and it is commercially
manufactured by hydrogenation of maleic anhydride to succinic
anhydride, followed by hydration to succinic acid. Recently major
efforts have been made to produce succinic acid by microbial
fermentation using a renewable feedstock. Many attempts have been
made to metabolically engineer the anaerobic central metabolic
pathway of Escherichia coli (E. coli) to increase succinate yield
and productivity. E. coli is extensively used in industry as a host
for many products due to the ease of genetic manipulation coupled
to its fast growth rate, standardized cultivation techniques and
cheap media. It is for this reason and for the need to produce
succinic acid economically at high concentrations and yields that
E. coli has been considered as a potential candidate to produce
this product of industrial interest.
[0006] It is well known that under anaerobic conditions and in the
absence of exogenous electron acceptors, E. coli metabolizes
glucose to a mixture of fermentative products consisting primarily
of acetate, ethanol, lactate and formate with smaller quantities of
succinate. NADH produced by the catabolism of glucose is
regenerated to NAD+ through the reduction of intermediate
metabolites derived from glucose in order to continue with
glycolysis. The distribution of products varies according to the
strain and growth conditions and is dictated by the way reducing
equivalents generated in the form of NADH are consumed so that an
appropriate redox balance is achieved by the cell.
[0007] Numerous efforts have been undertaken to make succinate the
major fermentation product in E. coli. Some genetic manipulations
previously studied are: deletion of the fermentative lactate
dehydrogenase (LDH) pathway, deletion of both the LDH and pyruvate
formate lyase (PFL) pathways and deletion of multiple pathways
including PFL and LDH pathways with an additional ptsG mutation
which restored the ability of the strain to grow fermentatively on
glucose and resulted in increased production of succinic acid.
Other studies include overexpression of phosphoenolpyruvate
carboxylase, (PEPC), overexpression of the malic enzyme and
overexpression of pyruvate carboxylase (PYC). Besides these genetic
manipulations, external means have been developed in order to
increase succinate production such as utilizing a dual phase
fermentation production mode which comprises an initial aerobic
growth phase followed by an anaerobic production phase, or by
changing the headspace conditions of the anaerobic fermentation
using carbon dioxide or hydrogen. It has been suggested that an
external supply of H.sub.2 might serve as a potential electron
donor for the formation of succinic acid, a highly reduced
fermentation product when compared to glucose.
[0008] Under fully anaerobic conditions, the maximum theoretical
yield (molar basis) of succinate from glucose is one based on the
number of reducing equivalents provided by this substrate. One mole
of glucose can provide only two moles of NADH, and two moles of
NADH can only produce one mole of succinate, therefore, in order to
surpass the maximum theoretical yield it is necessary to use part
of the carbon coming from glucose to provide additional reducing
power to the system.
[0009] Metabolic engineering has the potential to considerably
improve process productivity by manipulating the throughput of
metabolic pathways. Specifically, manipulating intermediate
substrate levels can result in greater than theoretical yields of a
desired product.
SUMMARY OF THE INVENTION
[0010] An aspect of the invention is directed toward a method of
increasing the production of succinate within a cell by mutating
one or more genes that encode enzymes involved in metabolic
reactions, and the mutations result in increased intracellular
levels of NADH.
[0011] Another aspect of the invention is directed toward a method
of increasing the NADH flux, in a cell, by mutating one or more
genes that encode enzymes involved in metabolic reaction, and the
mutations result in increased intracellular levels of NADH.
[0012] A further aspect of the invention is directed toward a
microorganism which contains one or more mutant genes, and displays
increased intracellular levels of NADH
BRIEF DESCRIPTION OF THE DRAWINGS
[0013] The accompanying drawings which are incorporated in and
constitute a part of this specification exemplify the invention and
together with the description, serve to explain the principles of
the invention:
[0014] FIG. 1 illustrates the central anaerobic metabolic pathway
of the strain SBS110MG showing inactivation of lactate
dehydrogenase and alcohol dehydrogenase pathways, and
overexpression of a plasmid that expresses pyruvate carboxylase
from Lactococcus lactis;
[0015] FIG. 2 illustrates the effect of formate supplementation (A)
on succinate yield and other metabolites (pyruvate (B); lactate
(C); acetate (D)) in the SBS110oP strain that is transformed with a
plasmid that expresses NAD-dependent formate dehydrogenase along
with a plasmid that expresses pyruvate carboxylase, and the strain
SBS110(pHL413) containing a plasmid that expresses pyruvate
carboxylase and a control plasmid pDHC30;
[0016] FIG. 3 illustrates metabolite concentrations (mM) and
product yields (succinate (B); formate (C); acetate (D); product
molar yield (E)) in anaerobic experiments using glucose as a carbon
source (A) in Luria Broth medium with a starting OD of 20.
Concentrations shown are from samples collected after 48 hours of
culture (average of triplicate cultures). The error bars represent
the standard deviation; and
[0017] FIG. 4 illustrates metabolite concentrations (mM) and
product yields (succinate (B); formate (C); acetate (D); succinate
molar yield (E)) in anaerobic experiments using glucose as a carbon
source (A) on LB medium with a starting OD of 4. Concentrations
shown are from samples collected after 48, 96 and 168 hours of
culture (average of triplicate cultures). The error bars represent
the standard deviation.
DESCRIPTION OF EXEMPLARY EMBODIMENTS
[0018] An embodiment of the invention is directed towards an E.
coli strain that is capable of achieving high succinate yield and
productivity by diverting maximum quantities of NADH for succinate
synthesis by striking a balance between cell physiology
requirements and achieving higher product yields.
[0019] An embodiment of the invention provides a strain of E. coli
in which both the ethanol and lactate synthesis pathways are
inactivated by mutating the genes, that code for the enzymes
involved in these pathways namely lactate dehydrogenase (LDH) and
alcohol dehydrogenase (ADH; AdhE). The AdhE protein of E. coli is
responsible for three different enzymatic functions. The ADH and
coenzyme A-linked acetaldehyde dehydrogenase (ACDH) functions are
involved in the conversion of acetyl-CoA to ethanol during
fermentation. Therefore, mutation of the adh gene entirely
inactivates the ethanol synthesis pathway of the respective mutant
strain.
[0020] An embodiment of the invention provides a double mutant
strain of E. coli in which the ldh and adh genes are inactivated
and the mutant strain is transformed with a plasmid expressing an
NAD-dependent formate dehydrogenase capable of NADH recycling.
[0021] In certain embodiments of the invention, the dual mutant E.
coli strain containing the plasmid expressing an NAD-dependent
formate dehydrogenase is further transformed with a plasmid
expressing the pyruvate carboxylase gene.
[0022] In an embodiment of the invention, the pyruvate carboxylase
gene is derived from Lactococcus lactis.
[0023] In other embodiments of the invention, the glucose permease
in the phosphotransferase (ptsG) system of the E. coli double
mutant is additionally mutated. This triple mutant is able to
further increase the molar succinate yield.
[0024] In certain embodiments of the invention, the native formate
dehydrogenase gene (fdhJ) is additionally mutated to generate a
triple mutant.
[0025] In an embodiment of the invention, an E. coli strain that
contains mutations in the adhE, ldh and ptsG genes is transformed
with a plasmid expressing the gene encoding pyruvate carboxylase.
In an embodiment of the invention, the pyruvate carboxylase gene is
derived from Lactococcus lactis.
[0026] In certain embodiments of the invention, an E. coli strain
that contains mutations in the adhE, ldh and fdhf genes is
transformed with a plasmid expressing the gene encoding pyruvate
carboxylase. In an embodiment of the invention, the pyruvate
carboxylase gene is derived from Lactococcus lactis.
[0027] In certain embodiments of the invention, increased succinate
yields are achieved by the increased conversion of pyruvate to
oxaloacetate by overexpressing phosphoenolpyruvate carboxylase
(PEPC) and/or pyruvate carboxylase (PYC).
[0028] In other embodiments of the invention, increased succinate
yields are achieved via reduced glucose uptake rate by using
glucose uptake deficient strains, such as a mutated ptsG
system.
[0029] In certain embodiments of the invention, increased succinate
yields are achieved via increased pyruvate to acetyl-CoA flux to
increase NADH supply by overexpressing an oxidoreductase enzyme
system or pyruvate formate lyase (PFL).
[0030] In other embodiments of the invention, the rate of succinate
formation can be further improved by using a dual phase process
where the growth and the production phase of the. culture are
operated in a sequential manner.
[0031] An embodiment of the invention is directed toward a
microorganism that contains one or more mutant genes and displays
increased yields of succinate.
[0032] Another embodiment of the invention is directed toward a
microorganism that displays a molar ratio of succinate yield to
glucose substrate of greater than 1.0.
[0033] A further embodiment of the invention is directed toward a
microorganism that displays a molar ratio of succinate yield to
glucose substrate that is at least about 1.3.
[0034] Another aspect of the invention is directed toward a
microorganism that displays a molar ratio of succinate yield to
glucose substrate that is greater than about 1.3.
[0035] Referring to FIG. 1, the central anaerobic metabolic pathway
of the strain SBS110MG showing inactivation of lactate
dehydrogenase and alcohol dehydrogenase pathways, and
overexpression of a plasmid that expresses pyruvate carboxylase
from Lactococcus lactis is depicted. The AdhE protein of E. coli is
responsible for three different enzymatic activities. Two of these
activities, ADH and coenzyme A-linked acetaldehyde dehydrogenase
(ACDH), are involved in the conversion of acetyl-CoA to ethanol
during fermentation
[0036] Referring to FIG. 2, two enzymes in the central anaerobic
pathway, lactate dehydrogenase (LDH) and alcohol dehydrogenase
(ADH; AdhE) were deactivated to generate the mutant strain SBS110.
A test mutant strain was created by transforming the dual mutant
strain with two plasmids, one expressing NAD-dependent formate
dehydrogenase (pASF2), and the other expressing pyruvate
carboxylase (pHL413). Control experiments were performed with the
dual mutant strain transformed with a plasmid expressing pyruvate
carboxylase (pHL413) and a control plasmid (pDHC30). The molar
succinate yield for the test mutant strain was higher (1.5 mol/mol)
than the control strain.
[0037] FIG. 3 illustrates the metabolite concentrations and product
yields in two mutant strains, a double mutant
(.DELTA.adhE.DELTA.ldhA) and a triple mutant
(.DELTA.adhE.DELTA.ldhA.DELTA.ptsG) transformed with a plasmid that
expresses pyruvate carboxylase (pHL413). The experimental details
and results are detailed below.
[0038] FIG. 4 illustrates the metabolite concentrations and product
yields in two triple mutant strains, SBS220MG
(.DELTA.adhE.DELTA.ldhA.DELTA.ptsG- ) and SBS880MG
(.DELTA.adhE.DELTA.ldhA.DELTA.fdhF) transformed with a plasmid that
expresses pyruvate carboxylase (pHL413). The experimental details
and results are detailed below.
EXAMPLE 1
Construction of Plasmids and Mutant Strains
[0039] Table 1 describes the strains used in this study and Table 2
describes the plasmids used in this study. Single mutations were
performed individually on MG1655 using the .lambda. Red recombinase
method of chromosomal disruption. Additional mutations were
introduced by P1-phage transduction with subsequent elimination of
the kanamycin resistance gene. Single gene disruption sites were
verified by PCR. Plasmid pHL413 contains the pyc gene from
Lactococcus lactis, which encodes the enzyme pyruvate carboxylase
that converts pyruvate to oxaloacetate.
1 TABLE 1 Strain Phenotype MG1655 Wild type (F.sup.-.lambda..sup.-)
SBS110 .DELTA.adhE.DELTA.ldhA SBS110MG .DELTA.adhE.DELTA.ldhA,
Km.sup.S SBS220MG .DELTA.adhE.DELTA.ldhA.DELTA.ptsG, Km.sup.S
SBS880MGK .DELTA.adhE.DELTA.ldhA.DELTA.fdhF, Km.sup.R SBS100MG
.DELTA.adhE, Km.sup.S CD55K .DELTA.ldhA, Km.sup.R SBS770MG
.DELTA.fdhF, Km.sup.R BW25113 .DELTA.ptsG, Km.sup.R
[0040]
2 TABLE 2 Plasmid Properties pHL413 Pyruvate carboxylase from
Lactococcus lactis cloned in pTrc99A, Ap.sup.R pASF2 NAD-dependent
formate dehydrogenase expression plasmid pTrc99A Control plasmid
pDHC30 Control plasmid
EXAMPLE 2
Culture of Bacterial Strain
[0041] Luria-Bertani (LB) broth medium supplemented with 200 mg/L
of 1:1:1 ampicillin, carbenicillin and oxacillin was used for all
aerobic cultivations. LB broth medium supplemented with 20 g/L of
glucose and 1 g/L of NaHCO.sub.3 was used for all anaerobic
cultivations and ampicillin was added at a concentration of 200
mg/L. Pyruvate carboxylase expression was induced by the addition
of isopropyl-.beta.-D-thiogalactopyranoside (IPTG) to a final
concentration of 1 mM.
[0042] A two-stage culture technique was used to examine the
accumulation of succinic acid in the culture broth. The first stage
comprises an initial aerobic growth phase followed by the second
stage, the anaerobic production phase. Cells were grown aerobically
in LB broth containing appropriate antibiotic concentration at
37.degree. C. and 250 rpm for 17 hours. Cells were harvested by
centrifugation and the supernatant discarded. Then the cells were
resuspended in fermentation medium at two different cell densities
of 4 or 20 OD units respectively. After resuspension, the cultures
were transferred aseptically to anaerobic culture containers, which
contained MgCO.sub.3. The containers were purged with CO.sub.2 at 1
L/min at STP.
[0043] For low inoculum experiments, triplicate cultures were grown
aerobically using 125-ml shake flasks containing 25 ml of LB medium
with appropriate antibiotic concentration. A volume of this culture
was centrifuged, and the cells collected were resuspended in 18 ml
of anaerobic medium to an initial OD of 4. The cells were
transferred aseptically to 45 ml glass anaerobic tubes containing
0.5 g of MgCO.sub.3. The resuspended culture was purged with
sterile CO.sub.2 at 1 L/min STP for 8 seconds and rapidly capped
with open top caps and PTFE/silicone rubber septa to ensure
anaerobic conditions. A sample of the initial media was saved for
analysis and samples were withdrawn with a syringe at 48, 96 and
168 h.
[0044] For higher inoculum experiments, aerobic cultures were grown
in a 2 L shake flask containing 400 ml of LB medium with
appropriate antibiotic concentration. A volume of this culture was
centrifuged, and the cells collected were resuspended in 10 ml of
anaerobic medium to an initial OD of 20. The cells were transferred
aseptically to 250 ml shake flasks containing 0.5 g of MgCO.sub.3.
The resuspended culture was purged with sterile CO.sub.2 at 1 L/min
STP for 1 min and rapidly capped with rubber stoppers to ensure
anaerobic conditions. For higher inoculum experiments, the use of
shake flasks allowed a larger CO.sub.2 liquid ratio avoiding
CO.sub.2 limitation conditions. A sample of the initial media was
saved for analysis and samples were withdrawn with a syringe at 24
and 48 h.
EXAMPLE 3
Analytical Techniques
[0045] Cell density was measured at 600 nm in a spectrophotometer.
Fermentation samples were centrifuged for 3 min at 13,000 g in a
microcentrifuge. The supernatant was filtered through a 0.45 .mu.m
syringe filter and stored chilled for HPLC analysis. The
fermentation products as well as glucose were quantified using a
Shimadzu HPLC system, equipped with a cation-exchanged column, a UV
detector and a differential refractive index detector. A mobile
phase of 2.5 mM H.sub.2SO.sub.4 solution at a 0.6 ml/min flow rate
was used and the column was operated at 55.degree. C.
EXAMPLE 4
Effects of Overexpression of Pyruvate Carboxylase
[0046] Experiments were performed with strain SBS110 transformed
with a plasmid expressing formate dehydrogenase along with a
plasmid expressing pyruvate carboxylase. As shown in Table 3 and
FIG. 2, an increased yield of succinate is observed when the strain
is grown in the presence of added formate.
3 TABLE 3 Formate added Metabolite yield 0 mM 100 mM Succinate
Yield SBS110(pDHC30 + pHL413) 1.18 1.44 SBS110(pASF2 + pHL413) 1.19
1.49 Pyruvate Yield SBS110(pDHC30 + pHL413) 0.39 0.74 SBS110(pASF2
+ pHL413) 0.51 0.91 Acetate Yield SBS110(pDHC30 + pHL413) 0.17 0.09
SBS110(pASF2 + pHL413) 0.08 0.07 Lactate Yield SBS110(pDHC30 +
pHL413) 0.01 0.02 SBS110(pASF2 + pHL413) 0.03 0.02 Ethanol
SBS110(pDHC30 + pHL413) BDL BDL SBS110(pASF2 + pHL413) BDL BDL BDL:
below detection level
[0047] Anaerobic tube experiments were performed under a complete
atmosphere of CO.sub.2 using an initial OD of 4 with strain
SBS110MG with and without plasmid pHL413 to assess the effect of
overexpressing the pyc gene. Samples taken at different time
intervals (48, 96 and 168 hrs) indicated that the expression of the
pyc gene (plasmid pHL413) was necessary to increase the glucose
uptake and to obtain high succinate yields. Fermentations with
SBS110OMG(pTrc99A) and SBS110OMG(pHL413) were terminated after 168
h. At this point the control strain consumed only 11% of the
initial glucose added (20 g/L) with low succinate yield and high
acetate yield while SBS110MG(pHL413) consumed 100% of the initial
glucose achieving a succinate yield of 1.3 mol/mol.
[0048] The effect of inoculum size on succinate production was also
examined by using a higher inoculum of 20 OD units. FIG. 3 shows
the results of these experiments, including glucose consumed, the
concentration of the metabolites produced and the product yields
after 48 h of culture.
[0049] A comparison of the results for SBS110MG (pTrc99A) and
SBS110MG(pHL413) shows the effect of overexpressing pyc on the
metabolic patterns of SBS110MG (FIG. 3). The glucose consumption
increased 4 fold; the succinate increased 25 fold from 5 mM to 132
mM from an initial glucose concentration of 104 mM. As expected,
overexpression of pyc increased the succinate yield from 0.2
mol/mol to 1.3 mol/mol, while the acetate yield dropped from 1.2
mol/mol to 0.8 mol/mol. The residual formate yield was also lower
in the strain overexpressing pyc relative to the control strain.
The residual formate dropped from 0.7 mol/mol to 0.5 mol/mol.
EXAMPLE 5
Effects of Deletion of Formate Dehydrogenase
[0050] To investigate the effect of eliminating the fdhF gene,
which encodes the native formate dehydrogenase (FDH) FDH-H, subunit
of the formate hydrogen lyase (FHL) complex that converts formate
to CO.sub.2 and H.sub.2, strain SBS880MGK was constructed by
eliminating the native fdhF gene from SBS110MG, both strains were
transformed with pHL413 and anaerobic tube experiments were
performed.
[0051] FIG. 4 shows the results obtained in anaerobic tube
experiments performed using an initial OD of 4. The cultures were
analyzed after different time intervals (48, 96 and 168 h). A
comparison of the results for the strain SBS110MG(pHL413) with
SBS880MGK(pHL413) indicates the effect of eliminating the native
FDH on the metabolic pattern of SBS110MG(pHL413). As can be seen
from FIG. 4, no significant differences were observed for the first
48 h of culture between both strains in glucose consumption,
succinate, acetate, residual formate levels or succinate yield.
After 96 h significant differences in glucose consumption and
succinate levels were noticed but no apparent change in the
succinate yield was observed. After 168 h a decrease in residual
formate yield was observed for SBS110MG(pHL413) relative to 48 h,
while the residual formate yield remain constant for the strain
lacking FDH activity. Glucose consumption, succinate levels and
yield were significantly lower at this time interval. The acetate
levels were similar, however the acetate yield was found to be
higher for the fdhf strain. Strain SBS110MG(pHL413) consumed 100%
of the glucose after 168 h while SBS880MGK(pHL413) consumed 62% of
the initial glucose. SBS110MG(pHL413) was able to sustain the
succinate molar yield in the range of 1.2 to 1.3 through the entire
fermentation period, while the succinate yield of SBS880MGK(pHL413)
dropped to 0.9 mol/mol by the end of the fermentation process.
[0052] After 48 hours of culture, succinate, residual formate or
acetate yield were similar in strains SBS110MG(pHL413) and
SBS880MGK(pHL413). After 96 h, acetate and residual formate yields
decrease for SBS110MG(pHL413) relative to SBS880MGK(pHL413),
however the succinate yield was not significantly different. After
168 h, the residual formate and acetate yield of SBS110MG(pHL413)
decreases abruptly to 0.19 and 0.8 respectively with concomitant
increase in glucose consumption and succinate levels (see FIG. 4)
in contrast to the strain lacking the native FDH. As expected the
residual formate yield remained unchanged after each time interval
analyzed with the fdhF strain.
EXAMPLE 6
Effect of PTSG Deletion
[0053] It has been shown that when a mutation of the ptsG was
introduced into E. coli strains that could ferment glucose, the
resulting strain was able to produce more succinate and less
acetate. Based on these findings and to evaluate the possibility of
a further increase in succinate yield and a decrease in acetate we
transferred the ptsG mutation into strain SBS110MG to create
SBS220MG. The triple mutant was transformed with plasmid pHL413 and
experiments were performed under anaerobic conditions using a high
cell density inoculum. The results of these experiments are
depicted in FIG. 3, including glucose consumed (mM) and the
concentration of different metabolites produced (mM) after 48 h of
culture. Pyruvate, lactate and ethanol concentrations were not
detected. Inactivation of the ptsG system significantly decreased
the glucose consumed, while increasing the succinate yield and
reducing the acetate yield as expected. The percent
increase/decrease in product yields of the ptsG strain relative to
the double mutant strain SBS110MG(pHL413) was a 7% increase in
succinate yield and a 15% decrease in acetate yield. The results
presented in FIG. 3 are the cultures analyzed after 48 h, but
additional runs performed with strain SBS110MG(pHL413) revealed
that 100% of the initial glucose could be consumed in 24 h. These
results indicate that the presence of the ptsG mutation slows down
the glucose consumption rate, therefore favoring succinate
generation and reducing the amount of acetate wasted.
* * * * *